The isolation of specific binding molecules (e.g., organic molecules) is a central problem in chemistry, biology and pharmaceutical sciences. For example, the vast majority of the drugs approved by the U.S. Food and Drug Administration are specific binders of biological targets which fall into one of the following categories: enzymes, receptors or ion channels. The specific binding to the biological target is not per se sufficient to turn a binding molecule into a drug, as it is widely recognized that other molecular properties (such as pharmacokinetic behaviour and stability) contribute to the performance of a drug. However, the isolation of specific binders against a relevant biological target typically represents the starting point in the process which leads to a new drug [Drews J. Drug discovery: a historical perspective. Science (2000) 287:1960-1964].
The ability to rapidly generate specific binders against the biological targets of interest would be invaluable also for a variety of chemical and biological applications. For example, the specific neutralization of a particular epitope of the intracellular protein of choice may provide information on the functional role of this epitope (and consequently of this protein). In principle, the use of monoclonal antibodies specific for a given epitope may provide the same type of information [Winter G, Griffiths A D, Hawkins R E, Hoogenboom H R. Making antibodies by phage display technology. Annu Rev Immunol. (1994) 12:433-455]. However, most antibodies do not readily cross the cell membrane and have to be artificially introduced into the cell of interest. In principle, intracellular antibodies can also be expressed into target cells by targeted gene delivery (e.g., by cell transfection with DNA directing the expression of the antibody). In this case, the antibody often does not fold, as the reducing intracellular milieu does not allow the formation of disulfide bonds which often contribute in an essential manner to antibody stability [Desiderio A, Franconi R, Lopez M, Villani M E, Viti F, Chiaraluce R, Consalvi V, Neri D, Benvenuto E. A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold. J Mol. Biol. (2001) 310: 603-615]. High affinity binding molecules amenable to chemical synthesis may provide a valuable alternative to antibody technology.
In Chemistry and Materials Sciences, the facile isolation of specific binding molecules may be useful for purposes as diverse as the generation of biosensors, the acceleration of chemical reactions, the design of materials with novel properties, the selective capture/separation/immobilization of target molecules.
The generation of large repertoires of molecules (e.g., by combinatorial chemistry; Otto S, Furlan R L, Sanders J K. Dynamic combinatorial chemistry. Drug Discov Today. (2002) 7: 117-125), coupled to ingenious screening methodologies, is recognized as an important avenue for the isolation of desired binding specificities. For example, most large pharmaceutical companies have proprietary chemical libraries, which they search for the identification of lead compounds. These libraries may be as large as >1 million members and yet, in some instances, not yield the binding specificities of interest [Balm H J, Stahl M. Structure-based library design: molecular modeling merges with combinatorial chemistry. Current Opinion in Chemical Biology (2000) 4: 283-286]. The screening of libraries containing millions of compounds may require not only very sophisticated synthetic methods, but also complex robotics and infrastructure for the storage, screening and evaluation of the members of the library.
The generation of large macromolecular repertoires (e.g., peptide or protein libraries), together with efficient biological and/or biochemical methods for the identification of binding specificities (such as phage display [Winter, 1994], peptides on plasmids [Cull M G, Miller J F, Schatz P J. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc Natl Acad Sci USA. (1992) 89: 1865-1869] ribosome display [Schaffitzel C, Hanes J, Jermutus L, Pluckthun A. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J Immunol Methods. (1999) 231: 119-135] yeast display [Boder E T, Wittrup K D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. (1997) 15: 553-557], periplasmic expression with cytometric screening [Chen G, Hayhurst A, Thomas J G, Harvey B R, Iverson B L, Georgiou G. Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS). Nat Biotechnol. (2001) 19:537-542], iterative colony filter screening [Giovannoni L, Viti F, Zardi L, Neri D. Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening. Nucleic Acids Res. (2001) 29: E27] etc.) may allow the isolation of valuable polypeptide binders, such as specific monoclonal antibodies, improved hormones and novel DNA-binding proteins. In contrast to conventional chemical libraries, protein libraries in the embodiments mentioned above may allow the efficient screening of as many as 1-10 billion individual members, in the search of a binding specificity of interest. On one hand, the generation of libraries of genes (e.g., the combinatorial mutagenesis of antibody genes; Winter, 1994; Viti F, Nilsson F, Demartis S, Huber A, Neri D. Design and use of phage display libraries for the selection of antibodies and enzymes. Methods Enzymol. (2000) 326:480-505) can directly be translated into libraries of proteins, using suitable expression systems (e.g. bacteria, yeasts, mammalian cells). Furthermore, methods such as phage display produce particles in which a phenotype (typically the binding properties of a protein, displayed on the surface of filamentous phage) is physically coupled to the corresponding genotype (i.e., the gene coding for the protein displayed on phage) [Winter, 1994], allowing the facile amplification and identification of library binding members with the desired binding specificity.
However, while biological/biochemical methods for the isolation of specific binding biomacromolecules can provide very useful binding specificities, their scope is essentially limited to repertoires of polypeptides or of nucleic acids [Brody E N, Gold L. Aptamers as therapeutic and diagnostic agents. J Biotechnol. (2000) 74:5-13]. For some applications, large biomacromolecules (such as proteins or DNA) are not ideal. For example, they are often unable to efficiently cross the cell membrane, and may undergo hydrolytic degradation in vivo.
In an attempt to mimic biological/biochemical methods for the identification of organic molecules with desired binding properties, out of a chemical library, Brenner and Lerner [Brenner S, Lerner R A. Encoded combinatorial chemistry. Proc Natl Acad Sci USA. (1992) 89: 5381-5383] have proposed the use of encoded chemical libraries (ECL). In their invention, the authors conceived a process of alternating parallel combinatorial synthesis in order to encode individual members of a large library of chemicals with unique nucleotide sequences. In particular, the authors postulated the combinatorial synthesis of polymeric chemical compounds on a solid support (e.g., a bead), where a step in the combinatorial synthesis would be followed by the synthesis (on the same bead) of a DNA sequence, to be used as a “memory tag” for the chemical reactions performed on the bead. In typical applications, DNA-encoded beads would be incubated with a target molecule (e.g., a protein of pharmaceutical relevance). After the DNA-tagged bead bearing the polymeric chemical entity is bound to the target, it should be possible to amplify the genetic tag by replication and use it for enrichment of the bound molecules by serial hybridization to a subset of the library. The nature of the polymeric chemical structure bound to the receptor could be decoded by sequencing the nucleotide tag.
The ECL method has the advantage of introducing the concept of “coding” a particular polymeric chemical moiety, synthesized on a bead, with a corresponding oligonucleotidic sequence, which can be “read” and amplified by PCR. However, the ECL method has a number of drawbacks. First, a general chemistry is needed which allows the alternating synthesis of polymeric organic molecules (often with different reactivity properties) and DNA synthesis on a bead. Second, the synthesis, management and quality control of large libraries (e.g., >1 million individual members) remains a formidable task. In fact, the usefulness of the ECL method has yet to be demonstrated with experimental examples.